Dual-Color Fluorescent Labeling of G Protein-Coupled Receptors

Abstract

Single-molecule FRET (smFRET) has become a powerful tool for biophysical and biological research. Currently, progress is limited by the absence of general methods to incorporate two different fluorophores at specific sites in proteins. Thus it would be of great value to develop a labeling scheme that enables two different fluorophores to be conjugated to one receptor (protein) in a well-defined manner. To prepare the dual-color labeled receptor two requirements need to be satisfied: 1) the generation of two different reactive handles at specific positions in the receptor and 2) the appropriate bioorthogonal labeling chemistries to distinguish between them. Here we show that the combination of cysteine/maleimide reaction and azido/alkyne cycloaddition (‘click’ chemistry) meets these conditions. Maleimide chemistry specifically targets at the thiol group of cysteine, which can be easily generated or removed by mutagenesis. In contrast, the click chemistry requires the azido or alkynyl functionality, which are not naturally present in proteins. We utilized the amber codon suppression technique to incorporate unnatural amino acids carrying these reactive groups. The first fluorophore is then attached using a maleimide handle and the second by a click reaction. In a series of proof-of-concept experiments utilizing the wellcharacterized visual photoreceptor rhodopsin, we have successfully demonstrated site-specific double labeling of a GPCR. Moreover, we have optimized the maleimide labeling protocol for rhodopsin and are conducting a comparative study on the copper-catalyzed click chemistry and copper-free click chemistry. We will discuss the results in terms of background level, material cost, and labeling stoichiometry. We envision a series of interesting applications for the dual-color labeled receptors, e.g., to probe the conformational change involved in GPCR activation. We propose to generalize this approach to other proteins, thereby permiting study of GPCR signalosome.